SMA is an autosomal recessive disorder characterized by degeneration of motor neurons in the spinal cord and skeletal muscle atrophy. SMA is the leading genetic cause of death in infancy for which no effective treatment is currently available. Although decreased levels of the survival motor neuron (SMN) protein cause SMA and inversely correlate with disease severity in both human patients and mouse model, the molecular mechanisms of SMA are unknown. SMN interacts directly with Sm and LSm proteins-an evolutionarily conserved family of proteins with diverse roles in RNA metabolism-and mediates their specific association with small nuclear RNAs into ribonucleoprotein complexes (snRNPs) that function in pre-mRNA processing. However, SMN activity in snRNP assembly does not explain the selective degeneration of motor neurons observed in SMA. Our hypothesis is that SMN role in RNP assembly may extend to other cellular noncoding RNAs (ncRNAs), possibly with a motor neuron-specific expression profile, and that some of these RNAs are additional yet-to-be-identified targets of Sm/LSm proteins. This scenario is suggested by the versatility of Sm/LSm proteins in forming different heteromeric complexes with distinct RNA binding characteristics. Defects in the biogenesis and function of these novel ncRNA targets of SMN may represent the disease trigger of SMA. We will take advantage of the capacity to generate large numbers of motor neurons differentiated from mouse ES cells to explore this possibility in the cell type that is affected in SMA. We will characterize the repertoire of ncRNAs that associate with SMN and Sm/LSm proteins as well as carry out an initial assessment of the potential involvement of the novel ncRNAs in SMA pathology. First, ncRNAs will be isolated from whole cell extracts by immunoprecipitation with specific monoclonal antibodies. Then, cDNA libraries will be generated from each immunoprecipitate and comprehensive identification of ncRNAs will be performed using high-throughput sequencing technologies. Following bioinformatic analysis with stringent filter criteria, selected ncRNAs will be analyzed for expression and association with SMN and Sm/LSm proteins by independent validation methods including quantitative RT-PCR. We will employ the same approach to investigate whether SMN deficiency-the disease trigger of SMA-affects the metabolism of validated ncRNAs. To do so, we will compare ncRNA expression and assembly into Sm/LSm-containing RNPs in cells with normal and reduced SMN. By carrying out qualitative and quantitative assessment of potential changes in the metabolism of ncRNAs in two distinct cell types (ES cells and ES cell-derived motor neurons) and two different physiological conditions (normal and low SMN levels), our study will reveal both ubiquitous and cell type-specific ncRNA targets of SMN and highlight their possible connection to SMA pathogenesis. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA) is a devastating motor neuron disease caused by decreased levels of the SMN protein. SMN plays a critical role in multiple aspects of RNA metabolism. We will carry out a comprehensive analysis of the repertoire of RNA targets of SMN with the goal of identifying novel RNA pathways that are regulated by SMN and whose disruption may contribute to motor neuron degeneration. These studies have the potential to provide unanticipated insights into the molecular mechanisms of SMA and to suggest new avenues of therapeutic intervention.